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Effects of adding salt to the diet of Asian sea bass Lates calcarifer reared in fresh or salt water recirculating tanks, on growth and brush border enzyme activity
Aquaculture 248 (2005) 315 – 324 www.elsevier.com/locate/aqua-online
Effects of adding salt to the diet of Asian sea bass Lates calcarifer reared in fresh or salt water recirculating tanks, on growth and brush border enzyme activity Sheenan Harpaza,T, Yaniv Hakima, Tatiyana Slosmana, O. Tufan Eroldogana,b a
Department of Aquaculture, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel b Department of Aquaculture, Faculty of Fisheries, Cukurova University, Bacali-Adana 01330, Turkey Received 15 July 2004; received in revised form 30 September 2004; accepted 1 March 2005
Abstract The Asian sea bass is a carnivorous, euryhaline species originating in seawater. It is currently being reared under intensive conditions in fresh and brackish water recirculating ponds. The effects of feeding sea bass a diet containing different levels of salt (NaCl) were evaluated in closed recirculating tanks in either fresh or salt (20x) water rearing conditions. Under freshwater conditions, the addition of salt to the diet resulted in a significant improvement in the feed conversion ratio (FCR), and had no effect on carcass composition of the fish. Under saltwater (20x) rearing conditions, it had no affect on either growth or FCR. The addition of salt enhanced the activity of the brush border enzymes alkaline phosphatase, lactase, and, to some extent, leucine amino peptidase in fish reared in freshwater, with the most pronounced effect exhibited in the pyloric caeca. The overall activity level of the brush border enzymes of fish reared in saltwater was found to be higher than that of the fish reared in freshwater. Fish reared in saltwater exhibited significant higher enzymatic activity of maltase, sucrase, and g-glutamyl transpeptidase when fed a diet with added salt compared to the control treatment. The results show that the addition of salt to the diet of Asian sea bass reared in freshwater at a level of up to 4% leads to better feed utilization coupled with a reduction in feed cost. D 2005 Elsevier B.V. All rights reserved. Keywords: Lates calcarifer; Asian sea bass; Dietary salt; Brush border enzymes
1. Introduction
T Corresponding author. Tel.: +972 3 9683388; fax: +972 3 9605667. E-mail address: [email protected] (S. Harpaz). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.03.007
The Asian sea bass, also known as Barramundi, is a carnivorous, euryhaline species originating in seawater, but which can be reared in freshwater and at high densities. It can be cultured in a variety of production systems including cages, and flow-through
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and closed recirculating production units. Barramundi are currently being explored as potential candidates for such systems, using marine water, brackish water, or freshwater. In freshwater-adapted fish, the passive outward flux of ions such as Na+ and Cl from the fish to the external medium, via the gills, faeces, and renal system, must be overcome by active uptake of ions (e.g., Na+, Cl, K+, and Ca2+) from the water and/or from the diet (Smith et al., 1989; Schmidt-Nielsen, 1997). Therefore, the diet constitutes an important source of salts that can satisfy the osmoregulatory requirements of fish kept in freshwater and thus spare energy used for osmoregulation, leaving more energy available for growth (Zaugg et al., 1983; Gatlin et al., 1992). It has been shown that the salinity of the rearing water has an influence on feed intake in rainbow trout. In salinities higher than freshwater (up to 28x), feed intake increased but the growth rate decreased, negatively affecting feed conversion ratio (MacLeod, 1977, 1978; Jurss et al., 1985). Yet, in euryhaline species, salinity affects growth in various ways and maximal growth is not always obtained under isosmotic conditions (Brett, 1979). Many researchers have addressed the issue of using dietary salt to help alleviate the problems associated with transferring salmonids to seawater or saltwater growing conditions (Shehadeh and Gordon, 1969; Zaugg and McLain, 1969; Potts et al., 1970; Basulto, 1976; Zaugg et al., 1983; Salman and Eddy, 1990; Pelletier and Besner, 1992). The results of these studies show a marked advantage in the use of dietary salt resulting in better survival. Even the transfer of tilapia, a freshwater (non-anadromous) species, to saltwater (15–20x) conditions was easier for fish that were pre-acclimatized by adding salt to their diet (Fontainhas-Fernandes et al., 2001). Zaugg and McLain (1969) reported that adding salt to the diet of fish has several advantages: it increases appetite and also acts as a humectant by reducing water activity. The affect on fish growth is not clear. Some studies have shown a positive effect (Gatlin et al., 1992; Fontainhas-Fernandes et al., 2000; Nandeesha et al., 2000; Eroldogan, 2003); some have not found this treatment advantageous (Shaw et al., 1975; Murray and Andrews, 1979); and others have reported that growth was hampered (Salman and Eddy, 1988). Zaugg and McLain (1969) found that in young coho
salmon, even the addition of only 1.5% salt resulted in a 7% weight gain reduction. Tacon and De Silva (1983) surveyed the mineral composition of various fish feeds available on the market and found that the content of salt in these feeds was on the average 1.5% but could be as high as 6% especially in the feeds used for early stage fingerling salmon. A large percentage of the dietary salt in commercial feeds for carnivorous fish originates from the fish meal component of the diet (Murray and Andrews, 1979). It is therefore important to take this into account when replacing fish meal with various plant-derived meals, which are not as rich in salt. In the Asian sea bass, the effects of different diet components (protein:energy ratio; carbohydrates) on growth were studied by Catacutan and Coloso (1995, 1997); kinematics of eating by Dowling et al. (2000); and effects of starvation and growth compensation by Tian and Qin (2003). The results of these studies show that the Asian sea bass is very similar to other carnivorous fish in its nutritional requirements. Yet, the effects of salt addition to the diet of this fish have not been studied. Since the Asian sea bass originates in seawater, we decided to explore the advantages of adding salt (NaCl) to the fish diet. Better utilization of the food is dependent on both its digestion and absorption. This process is a result of the total activity of digestive enzymes, including the final stages of digestion and absorption which take place in the brush border section of the intestines. The availability of the enzymes and their ability to transfer the digested food through the epithelial luminal membrane of the intestine at that stage are crucial (Klein et al., 1998). The goals of the present study were: to explore the effects of adding different levels of dietary salt to the extruded pelletized diet feed of Asian sea bass; to monitor the fish growth and feed conversion ratio in both freshwater and saltwater; and to study the effects of these treatments on the activity of the brush border digestion enzymes: sucrase, maltase, lactase, g-glutamyl transpeptidase, and leucine-amino peptidase. This was done in three separate sections of the digestive tract, namely, the pyloric caeca, and the upper and lower intestines. Since the final stages of the digestion and absorption are carried out by these enzymes, it is essential to explore the effects of the different treatments on their activity.
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2. Materials and methods
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adding freshwater or saltwater in the manner described above.
2.1. Experimental tanks and rearing conditions 2.2. Fish Six plastic tanks with conical bottoms, each containing 250 l, were connected to a central biofilter and settling unit of 350 l, through which the water was circulated with the aid of a submersible pump, at a rate equivalent to total tank replacement every 1.5 h. The bio-filter contained small plastic cylinders, 1.5 cm long with a diameter of 1 cm, kept inside plastic net bags to facilitate cleaning/rinsing. The experimental set up had two such units, totaling 12 tanks altogether. The tank water was heated using electric heating elements (2 kW A) immersed in each of the settling tanks and connected to a thermostat to ensure a constant temperature of 30 8C F1 8C throughout the experiment. The tanks were set indoors and the light source was natural photoperiod enhanced with florescent light, providing a light intensity of 1200 lx during the day hours. A belt feeder situated above each tank provided a small shaded area. Water quality parameters were monitored three times a week and did not deviate from standard conditions considered favorable for these fish (i.e., an oxygen level of 90–100% saturation, average ammonia (measured as NH4+) level of 0.42 mg/l, and an average nitrite level of 1 mg/l). In the saltwater phase, the values were higher and reached an average of 0.84 and 7.75 mg/l, respectively. Ammonia and nitrite levels were determined using Aquamerck kits for fresh and saltwater. Each day, the temperature was registered using a submersed maximum/minimum thermometer; dead fish (if occurred) were removed and the settling tank was cleaned by draining its stand pipe, thus allowing new water to compensate for the water loss during this brief flushing (amounting to approximately 2% of the water volume). Every 3–4 days, a more thorough cleaning of the settling tank was carried out as well as flushing of the organic particles from the biofilter. The amount of water replaced during the course of this procedure was roughly 5%. The water replaced during the saltwater phase of the experiment was a mixture of marine salt and freshwater. The salinity level was monitored every 2–3 days using a refractometer and adjusted by
Postlarvae Asian sea bass (Lates calcarifer) were purchased from Thailand, acclimatized to freshwater at an early stage (postlarvae of less than 0.3 g), and reared under indoor conditions at our laboratory. Fish were selected when they reached an average weight of around 110 g. Prior to the beginning of the experiment, the fish were anesthetized by adding clove oil mixed with ethanol to the tank water. Individual numbered tags were inserted into their caudal muscle and they were weighed using an electronic balance. The experimental tanks were each stocked with a total of 10 fish. The average initial weight of the fish participating in the experiment was 107.22F8.44 g and there were no significant differences among the experimental tanks at the onset of the experiment ( P N 0.05). During the freshwater phase, all the fish in each tank were individually weighed and counted once every fortnight. This was done in order to monitor survival and to update the feeding ration, according to the growth rate in each individual tank. In addition, the food ration was adjusted every other week according to predicted growth. At the end of the freshwater phase, the fish were acclimatized to saltwater by increasing the salinity of the rearing water by 2x per day over a period of 10 days. At the beginning of the saltwater (20x) phase, each tank had seven fish with an initial average weight of 182.11F22.70 g. The fish were individually weighed at the beginning and end of this phase and feeding was adjusted every week according to the predicted growth. The fish were weighed using a digital balance (Swiss quality, Precisa 1000-3000 D) after drying them briefly on tissue paper to absorb excess water. 2.3. Treatments tested 2.3.1. Feed preparation Feed was in the form of sinking extruded pellets— 3 mm in diameter, containing 48% protein, 12% fat, 1.6% fiber, and 1.4% NaCl, and manufactured for carnivorous fish by Raanan Marketing (RMC Ltd., Israel). NaCl-enriched diets were prepared by adding
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the NaCl to the pellets (above the basal level) and spray coating them with a mixture of NaCl and poultry oil to prevent leaching of the salt to the water after immersion. This resulted in a proportional reduction of the ration ingredients replaced by the salt. Preliminary evaluation showed that by using this method, the mineral level in the pellets was not significantly reduced following immersion in water for a period comparable to the time the pellets were consumed by the fish. Proximate analysis of the prepared pellets performed before the beginning of the experiment revealed that the level of ash (corresponding to mineral levels) was proportionally higher in the diets that had salt added to them. 2.3.2. Tested diets A. Feed pellets containing no additional NaCl (control = commercial feed used for predatory fish which has 48% protein, 12% fat, 1.6% fiber, and 1.4% NaCl). B. Feed pellets (as in A) containing 1% additional NaCl. C. Feed pellets (as in A) containing 2% additional NaCl. D. Feed pellets (as in A) containing 4% additional NaCl. Each of the above treatments was tested in three replicates and since two groups of tanks were used, each of the treatments was represented in each group of tanks. According to the feeding charts for Asian sea bass similar in size to the ones used in the present study and kept at a temperature of around 30 8CF1 8C, the feeding rate should be around 2% of the biomass per day. At the onset of the experiment, a ration of 1.5% was used and from 1 week onwards, it was updated to the level of 2% of the biomass per day. Fish were fed 7 days a week with the aid of a belt feeder, which delivered the ration over a period of 6 h during the light phase of the day. The duration of the freshwater testing phase was 50 days, and that of the saltwater (20x) one was 25 days. At the end of the experiment, all the fish in all the tanks were individually weighed in order to compare the effects of the treatments on growth, feed conversion ratio (i.e., total feed given divided by weight gain (FCR)), and survival.
Specific growth rate (SGR) was calculated in the following manner: SGR ¼
ln final weight ln initial weight days of growth
100
2.3.3. Body composition analysis Samples were ground using a meat grinder and thoroughly mixed. Samples of each fish were then taken for analysis as follows: the level of protein was determined according to the Kjeldahl procedure and by the 2000 Digestion System (Tekator, Sweden). Fat was determined using a Soxhelt extractor with 95% ethanol. Ash levels were determined using a BIFATherm C-36 oven (BIFA, Ramat Gan, Israel) set at 600 8C. 2.4. Evaluation of brush border enzymes At the end of each phase of the experiment, three fish from each tank were sacrificed using a scalpel to sever the spine. The fish were taken prior to feeding (to ensure empty digestive tract). The entire digestive tract was removed and adipose tissue carefully cleaned off. When necessary, remaining food residues were gently squeezed out. The digestive system was then divided into the following sections: pyloric ceaca, upper intestines, and lower intestines (each comprising about 50% of the intestine). The dissected sections were stored in marked plastic containers in a 20 8C freezer and evaluated later according to Harpaz and Uni (1999) with slight modifications. Prior to evaluation, stored sections were thawed, transferred to glass tubes, and crushed using a homogenizer. Ten milliliters of distilled water was added to every milliliter of tissue and homogenized using a PRO Scientific Inc. model 200 homogenizer at a speed of 5 V. Between each of the different homogenizing processes, the instrument was thoroughly cleaned with distilled water and wiped with tissue paper. The tissues were kept on ice throughout the whole process. A series of preliminary tests was carried out to determine the appropriate dilutions to be used in the evaluation of the enzymatic activity (Felach, 2002).
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The thawed samples were diluted at a ratio of 1:10 and centrifuged with an Eppendorf 5810R centrifuge for 5 min at a speed of 13,500 g and at 4 8C. 2.4.1. Leucine-amino peptidase (LAP) The activity of LAP (LAP; EC 3.4.11.1) was measured using a kit manufactured by SPINREACT (catalog no. 1001250). One hundred and fifty microliters of reagent were added to 10 Al of the diluted homogenate, placed in an ELISA plate, and, after 5 min of incubation at room temperature, the sample results were read at 1 min intervals at a wave length of 405 nm using a Tecan Sunrise ELISA reader. Units of activity were determined using the following equation: LAPðU=mg PÞ ¼ ðð DA=min TV Þ=ðEF SV ÞÞ ð D=PÞ where: DA/min is total increment per minute; TV (160 Al) is the total reaction volume (homogenate + reagent); EF (1.167) is excitation coefficient of p-nitrophenoxide at 405 nm; SV is homogenate (10 Al); D is dilution (160 Al); and P is protein (mg) in the homogenate. 2.4.2. Alkaline phosphatase (AP) The activity of this enzyme (AP; EC 3.1.3.1) was measured using a kit manufactured by ThermoTrace (catalog no. TR11015). In this reaction, p-nitrophenoxide obtained is proportional to the enzymatic activity and has a distinct yellow color that can be quantified (Walsh, 1979). Two hundred microliters of reagent were added to 5 Al of the diluted homogenate placed in an ELISA plate and read as described for LAP. One unit of enzymatic activity was calculated as the quantity required in order to release 1 Amol of the product ( p-nitrophenoxide) per minute per milligram of protein. Units of activity were determined as described for LAP. Units are the same except for the EF coefficient, which is 18.8, the excitation coefficient of p-nitrophenoxide at 405 nm. 2.4.3. c-Glutamyl transpeptidase (c-GT) The activity of this enzyme (g-GT; EC 2.3.2.2) was measured using a kit manufactured by ThermoTrace
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(catalog no. TR19110). Two hundred microliters of reagent were added to 10 Al of the diluted homogenate placed in an ELISA plate, and read as described for LAP. One unit of enzymatic activity was calculated as the quantity required in order to release 1 Amol of the product (5-amino-2-nitrobenzoate) per minute per milligram of protein (Stump, 1955). Units of activity were determined using the same equation as described for LAP except for the EF coefficient, which is 9.5 for the 5-amino-2-nitrobenzoate produced in this reaction at 405 nm. 2.4.4. Sucrase maltase and lactase determination The activity of the enzymes sucrase (EC 3.2.1.48), maltase (EC 3.2.1.20), and lactase (EC 3.2.1.108) was determined using the following substrates: sucrose produced by Fluka (catalog no. 84099), maltose produced by Sigma (catalog no. M-5885), and lactose produced by Fluka (catalog no. 61340). The activity of these enzymes is as follows: sucrase
sucrose Y glucose þ fructose maltase
maltose Y glucose þ glucose lactase
lactose Y glucose þ galactose The presence of glucose, as a measure of enzymatic activity, was then determined using glucose color reagent no. TR15421 (ThermoTrace) and the reaction was compared to a LT00211.01 standard manufactured by the same company. Forty microliters of homogenate was mixed with 40 Al of substrate at a concentration of 0.056 M. The color reaction was then read at 340 nm using an Ultrospec 2000 Pharmacia Biotech spectrophotometer. Protein was determined using a kit based on the Bradford method and manufactured by Bio-Rad (catalog no. 500-0205). The activity was measured by mixing 20 Al of the homogenate with 1 ml of the reagent, and after 5 min at room temperature, the color intensity was read at 595 nm wave length, while comparing it to a standard (2 mg/ml BSA standard; Bio-Rad color reagent no. 500-0206).
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Statistical analysis was carried out using JMP statistical package. One- and two-way ANOVA followed by a Tukey–Kramer HSD test, set at P b 0.05 were conducted.
Table 2 Body composition of Asian sea bass reared in freshwater and fed diet containing varying levels of added salt Body Control 1% added composition (no added salt) salt Ash Protein Lipid
The growth results for both experiments are presented in Table 1. It can be seen that in the freshwater phase, the addition of salt to the diet resulted in a significantly better feed conversion ratio and a higher (yet not significant) growth rate. In the saltwater phase, the addition of salt to the diet did not result in an improvement of growth or feed conversion ratio. The survival rate in the freshwater phase was very high (97%) and all, except for one tank, had 100% survival. Survival in the saltwater phase was lower (90%) and the variability in growth (Table 1) in this phase was much higher. The body composition of the fish reared in freshwater and fed diets containing different levels of salt was not significantly different among the treatments, although there was a trend showing less fat accumulation in the fish fed diets containing additional salt (Table 2). 3.2. Enzymes activity A clear dose–response relationship between the level of added salt and activity level of alkaline phosphates and lactase was found in the pyloric caeca of fish reared in freshwater (Figs. 1 and 2). The Table 1 Results of growth trials in freshwater and saltwater environments, with Asian sea bass fed diets containing different levels of dietary salt Groups
FW
SW
FCR
SGR
FCR
SGR
Control 1% NaCl 2% NaCl 4% NaCl
1.77 F 0.044b 1.61 F 0.030a 1.60 F 0.023a 1.67 F 0.004ab
0.98 F 0.037 1.08 F 0.039 1.09 F 0.027 1.06 F 0.025
1.37 F 0.167 1.60 F 0.170 1.72 F 0.273 1.37 F 0.183
1.19 F 0.125 1.03 F 0.107 0.99 F 0.157 1.17 F 0.135
Values followed by different superscript letters are significantly different ( P b 0.05).
4% added salt
3.98 F 0.27 3.68 F 0.60 3.59 F 0.54 18.84 F 0.42 19.44 F 0.46 18.72 F 0.91 5.82 F 0.58 6.83 F 1.38 6.57 F 1.00
activity of leucine amino peptidase in the fish reared in freshwater was significantly higher in those fed the diet containing 1% additional salt, while the other levels of salt addition did not result in enhanced activity (Fig. 3). Activity of g-glutamyl transpeptidase was not enhanced by the addition of salt to the diet, but the overall activity in the lower intestine of fish reared in saltwater was significantly higher than that found in this section of the intestine in fish reared in freshwater (Fig. 4). In the fish reared in saltwater, the addition of salt significantly enhanced the activity of maltase, sucrase, and g-glutamyl transpeptidase compared to the con-
a LEVEL OF ACTIVITY (U / mgP)
3.1. Growth and survival
CONTROL 8 7 6 5 4 3 2 1 0
1%
2%
4%
a b b c
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
b LEVEL OF ACTIVITY (U / mgP)
3. Results
4.41 F 0.05 18.91 F 0.22 7.97 F 1.42
2% added salt
CONTROL
1%
2%
4%
9 8 7 6 5 4 3 2 1 0
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
Fig. 1. Activity of alkaline phosphatase (AP) measured in the different sections of the digestive tract of Asian sea bass reared in freshwater (a) and in 20x saltwater (b) and fed diets containing no added salt or 1%, 2%, and 4% added salt.
S. Harpaz et al. / Aquaculture 248 (2005) 315–324
CONTROL
500
1%
2%
4%
a
400
a 300
ab
200
b 100 0
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
CONTROL
1%
2%
4%
700 600 500 400 300 200 100 0
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
Fig. 2. Activity of lactase measured in different sections of the digestive tract of Asian sea bass reared in freshwater (a) and in 20x saltwater (b) and fed diets containing no added salt or 1%, 2%, and 4% added salt.
trol. The overall activity of the brush border enzymes was significantly higher in these fish compared with those reared in freshwater in all enzymes tested except for lactase and alkaline phosphatase (Table 3).
4. Discussion Smith et al. (1989) showed that the dietary sodium intake of salmonids kept in freshwater increased by about eightfold from winter to summer (equaling the branchial sodium influx). This corresponds to the increase in feeding and shows that almost all the sodium required can be derived from dietary salt. This can therefore be used as a source of salts for the fish kept in freshwater, providing ions which the fish cannot obtain from the hypotonic environment.
a LEVEL OF ACTIVITY (U / mgP)
LEVEL OF ACTIVITY (µ µmol / mg P / Hour)
b
The addition of salt to the diet of freshwater carp at a level of 1.5% resulted in significantly better growth and is in widespread use in India (Nandeesha et al., 2000). In an experiment conducted with juvenile red drum, it was clearly shown that the addition of 2% NaCl to the diet resulted, as was the case in our study, in greater feed efficiency and greater weight gain (Gatlin et al., 1992). In the same experiment, fish kept in brackish water of 5x and fed a salt-enhanced diet showed an increase in weight gain over the basal diet, but this increase was not significant. Tests conducted on red drum kept in seawater showed no advantage to the addition of NaCl at a level of 2% over the basal diet (Gatlin et al., 1992). The absorption of mono-valent ions increases as a result of the adaptation to the hyperosmotic medium (Shehadeh and Gordon, 1969). It seems that even though fish reared in saltwater were exposed to additional salt from their food, this quantity of salt is negligent compared to the overall amount of salt from the environment that the fish
CONTROL
1%
2%
4%
0.15
a
0.12
b 0.09
b
b
a a ab
0.06
b
0.03 0.00
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
b LEVEL OF ACTIVITY (U / mgP)
LEVEL OF ACTIVITY (µmol / mg P / Hour)
a
321
CONTROL
1%
2%
4%
0.25 0.20 0.15 0.10 0.05 0.00
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
Fig. 3. Activity of leucine amino peptidase (LAP) measured in the different sections of the digestive tract of Asian sea bass reared in freshwater (a) and in 20x saltwater (b) and fed diets containing no added salt or 1%, 2%, and 4% added salt.
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LEVEL OF ACTIVITY (U / mgP)
a
CONTROL
2%
4%
0.11 0.09 0.07 0.04 0.02 0.00
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
b LEVEL OF ACTIVITY (U / mgP)
1%
0.11
0.09
0.07
0.04
0.02
0.00
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
Fig. 4. Activity of g-glutamyl transpeptidase (g-GT) measured in the different sections of the digestive tract of Asian sea bass reared in freshwater (a) and in 20x saltwater (b) and fed diets containing no added salt or 1%, 2%, and 4% added salt.
absorb through the intestine and body surface (Potts et al., 1970). The results of our experiment show that fish reared in saltwater and fed feed containing additional salt exhibited a slightly lower (yet non significant) growth rate trend compared with the control group that had no added salt to their feed (Table 1). The enzymatic activity in these fish was different than that observed in freshwater (Figs. 1b, 2b, and 3b). Thus, even if the addition of salt did have a slight impact on growth, this effect is reduced due to better utilization of the food, which is manifested by the increase in the enzymatic activity. It should be mentioned that although the addition of salt did not result in better growth, it did not hamper the growth of the fish reared in saltwater. Therefore, it can be considered as advantageous since the diet containing additional salt in our experiment had a reduction in the level of other ingredients (nutrient dilution) corresponding to the level of added salt.
The overall activity of the brush border enzymes in fish reared in saltwater (20x) was found to be significantly higher than that in fish reared in freshwater. This is probably due to the fact that the Asian sea bass is a catadromous fish, which spends a substantial part of its life in marine water. The better enzymatic activity in the fish fed a diet containing added salt can be explained by the absorption mechanism of the end products—glucose and amino acids. Since the glucose and most of the amino acid absorption is depended on the Na+/K+ ATPase pump (Klein et al., 1998), a higher concentration of Na+ in the lumen might lead to a better absorption of carbohydrates and amino acids. Since the enzyme activity might be inhibited by its end product (carbohydrates or amino acids) (De la Fuente et al., 1997), reduction of the end product concentration can lead to better enzymatic activity in the lumen of fish fed feed enhanced with NaCl. Progressive transfer of rainbow trout to seawater resulted in enhancement of intestinal and branchial Na+/K+ ATPase activity (Fuentes et al., 1997). It is possible that the addition of salt to the feed acted in a similar manner as a bprogressiveQ transfer to salt conditions. Fish feed constitutes one of the most expensive components in the rearing of carnivorous fish, and the extremely high protein levels required for these fish (Wilson, 2002) are also the major source of nitrogenous products harmful to fish in closed recirculating systems. Therefore, the fact that the addition of salt to the diet of the fish reared in freshwater resulted in a better FCR is of great importance. The marked increase in the activity of the brush border enzymes, observed in the freshwater phase, especially in the pyloric caeca (Figs. 1 and 2)
Table 3 Overall activity of different brush border enzymes in Asian sea bass reared in freshwater or saltwater (20 x) medium Enzyme evaluated
Freshwater medium Saltwater medium
Maltase 2184.59 F 73.88 Sucrase 377.48 F 11.02 Lactase 225.96 F 23.07 Leucine amino peptidase 0.074 F 0.0027 g-GT 0.031 F 0.0013 Alkaline phosphatase 2.38 F 0.101 T Values are significantly different ( P b 0.05).
2590.67 F 77.70T 452.21 F16.70T 266.45 F 12.99 0.141 F 0.011T 0.04 F 0.0026T 2.52 F 0.359
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might explain the improved results. The pyloric caeca is emerging as one of the important parts of the digestive tract in carnivorous fish especially as far as the final digestion and absorption of food are concerned (Buddington and Diamond, 1987). Interestingly, in the fish reared in saltwater, the activity of g-glutamyl transpeptidase was much higher in the lower intestine compared with the activity exhibited in the fish held in freshwater and in all other sections of the digestive tract (Fig. 4). The lactase found in the fish intestines most probably originated from bacterial sources. The addition of salt to the diet resulted in an increase in the lactase activity level (Fig. 2), probably due to the altered conditions inside the fish reared in freshwater, which were more favorable for the lactase-supplying bacteria. The conditions in saltwater reared fish were not changed by the additional salt in the diet and therefore the activity of this enzyme in the fish reared in saltwater was not higher (Table 3). The significantly better activity of leucine amino peptidase that was found in the treatment of (1%) salt added to the feed compared with the control and the enhancement of the activity of this protease is of special importance since the Asian sea bass is a carnivorous fish. The presence and high level of activity of carbohydrate-related enzymes in the Asian sea bass have been reported (Sabapathy and Teo, 1993). Maltase is required for the breakdown of maltose produced during the hydrolysis of starch and was found in high levels in the pyloric caeca of Asian sea bass (Sabapathy and Teo, 1993). The intensive rearing of fish in closed recirculating systems is costly and calls for methods that will enhance food utilization while efficiently using the biological filter in the recirculating systems. Utilizing a diet enhanced with up to 4% salt has an advantage, as can be seen from the results obtained in the present study. It can lead to better feed utilization under intensive production conditions and can reduce the cost of feed since we are diluting expensive components with a cheap mineral. This simple method can also be applied by farmers and does not require special means. Total salt content in the diet should also be taken into account when using a plant substitute to replace fish meal, which is one of the major contributors of salt to the diet (Murray and Andrews, 1979).
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Acknowledgement This study was funded by a grant obtained from the European Commission (contract no. ICA3-CT2002-10001).
References Basulto, S., 1976. Induced saltwater tolerance in connection with inorganic salts in the feeding of Atlantic salmon (Salmo salar L.). Aquaculture 8, 45 – 55. Brett, J.R., 1979. Environmental factors and growth. In: Hoar, W.S., Randal, D.J., Brett, J.R. (Eds.), Fish Physiology, vol. VIII. Academic Press, NY, pp. 599 – 675. Buddington, R.K., Diamond, J.M., 1987. Pyloric caeca of fish: a dnewT absorptive organ. Am. J. Physiol. 252, G65 – G76. Catacutan, M.R., Coloso, R.M., 1995. Effect of dietary protein to energy ratios on growth, survival, and body composition of juvenile Asian seabass Lates calcarifer. Aquaculture 131, 125 – 133. Catacutan, M.R., Coloso, R.M., 1997. Growth of juvenile Asian seabass, Lates calcarifer, fed varying carbohydrate and lipid levels. Aquaculture 149, 137 – 144. De la Fuente, J.L., Rumbero, A., Martı´n, J.F., Liras, P., 1997. DELTA-1-piperideine-6-carboxylate dehydrogenase, a new enzyme that forms alpha-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes. J. Biochem. 327, 59 – 64. Dowling, N.A., Hall, S.J., Mitchell, J.G., 2000. Foraging kinematics of barramundi during early stages of development. J. Fish Biol. 57, 337 – 353. Eroldogan, O.T., 2003. Acclimation of European sea bass (Dicentrarchus labrax) to freshwater and determination of its optimal feeding rates in freshwater. Ph.D. thesis, University of Cukurova, Adana, Turkey. Felach, J., 2002. Effects of feeding regimes and salinity of water on the activity of intestinal enzymes of fish as a measure of efficiency of food absorption. Master of Science Thesis (Hebrew with English abstract). The Hebrew University, Jerusalem. Fontainhas-Fernandes, A., Monterio, M., Gomes, E., Reis-Henriques, M.A., Coimbra, J., 2000. Effect of dietary sodium chloride acclimation on growth and plasma thyroid hormones in tilapia Oreochromis niloticus (L.) in relation to sex. Aquac. Res. 31, 507 – 517. Fontainhas-Fernandes, A., Russell-Pinto, F., Gomes, E., ReisHenriques, M.A., Coimbra, J., 2001. The effect of dietary sodium chloride on some osmoregulatory parameters of the teleost, Oreochromis niloticus after transfer from freshwater to seawater. Fish Physiol. Biochem. 23, 307 – 316. Fuentes, J., Soengas, J.L., Rey, P., Rebolledo, E., 1997. Progressive transfer to seawater enhances intestinal and branchial Na+/K+ ATPase activity in non-anadromous rainbow trout. Aquac. Int. 5, 217 – 227. Gatlin III, D.M., MacKenzie, D.S., Craig, S.R., Neill, W.H., 1992. Effects of dietary sodium chloride on red drum juveniles in waters of various salinities. Prog. Fish-Cult. 54, 220 – 227.
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Harpaz, S., Uni, Z., 1999. Activity of intestinal mucosal brush border membrane enzymes in relation to the feeding habits of three aquaculture fish species. Comp. Biochem. Physiol. 124A, 155 – 160. Jurss, K., Bittorf, T.H., Vokler, T.H., 1985. Influence of salinity and ratio of lipid to protein in diets on certain enzyme activities in rainbow trout (Salmo gairdneri Richardson). Comp. Biochem. Physiol. 81B, 73 – 79. Klein, S., Cohn, S.M., Alpers, D.H., 1998. The alimentary tract in nutrition. In: Shils, M.E., Olson, A.J., Shike, M., Ross, A.C. (Eds.), Modern Nutrition in Health and Disease, ninth edition, Williams and Wilkins, Baltimore, MD, pp. 605 – 630. MacLeod, M.G., 1977. Effects of salinity on food intake, absorption and conversion in the rainbow trout, Salmo gairdneri. Mar. Biol. 43, 93 – 102. MacLeod, M.G., 1978. Relationships between dietary sodium chloride, food intake and food conversion in the rainbow trout. J. Fish Biol. 13, 73 – 78. Murray, M.W., Andrews, J.W., 1979. Channel catfish: the absence of an effect of dietary salt on growth. Prog. Fish-Cult. 41, 155 – 156. Nandeesha, M.C., Gangadhar, B., Keshavanath, P., Varghese, T.J., 2000. Effect of dietary sodium chloride supplementation on growth, biochemical composition and digestive enzyme activity of young Cyprinus carpio (Linn) and Cirrhinus mrigala (Ham). J. Aquac. Trop. 15, 135 – 144. Pelletier, D., Besner, M., 1992. The effects of salty diets and gradual transfer to sea water on osmotic adaptation, gill Na+–K+ ATPase activation, and survival of brood charr Salvelinus fontinalis Mitchell. J. Fish Biol. 41, 791 – 803. Potts, W.T.W., Foster, M.A., Stather, J.W., 1970. Salt and water balance in salmon smolts. J. Exp. Biol. 52, 533 – 564. Sabapathy, U., Teo, L.H., 1993. A quantitative study of some digestive enzymes in the rabbitfish, Siganus canaliculatus and the sea bass Lates calcarifer. J. Fish Biol. 42, 595 – 602. Salman, N.A., Eddy, F.B., 1988. Effect of dietary sodium chloride on growth, food intake and conversion efficiency in rainbow trout (Salmo gairdneri Richardson). Aquaculture 70, 131 – 144.
Salman, N.A., Eddy, F.B., 1990. Increased sea-water adaptability of non-smolting rainbow trout by salt feeding. Aquaculture 86, 259 – 270. Schmidt-Nielsen, K., 1997. Water and osmotic regulation. In: Schmidt-Nielsen, K. (Ed.), Animal Physiology Adaptation and Environment, fifth edition, Cambridge University Press, pp. 301 – 354. Shaw, H.M., Saunders, R.L., Hall, H.C., Henderson, E.B., 1975. Effect of dietary sodium chloride on growth of Atlantic salmon (Salmo salar). J. Fish. Res. Board Can. 32, 1813 – 1819. Shehadeh, Z.H., Gordon, M.S., 1969. The role of the intestine in salinity adaptation of the rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 30, 397 – 418. Smith, N.F., Talbot, C., Eddy, F.B., 1989. Dietary salt intake and its relevance to ionic regulation in freshwater salmonids. J. Fish Biol. 35, 749 – 753. Stump, P.K., 1955. g-Glutamyl transpeptidase. In: Meister, A. (Ed.), Methods in Enzymology, vol. 2. Academic Press, New York, pp. 263 – 272. Tacon, A.G.J., De Silva, S.S., 1983. Mineral composition of some commercial fish feeds available in Europe. Aquaculture 31, 11 – 20. Tian, X., Qin, J.G., 2003. A single phase of food deprivation provoked compensatory growth in barramundi Lates calcarifer. Aquaculture 224, 169 – 179. Walsh, C., 1979. Alkaline phosphatase. Enzymatic reaction mechanisms. In: Bartlett, A.C. (Ed.), Methods in Enzymology, vol. 62. Academic Press, New York, pp. 192 – 194. Wilson, R.P., 2002. Amino acids and proteins. In: Halver, J.E., Hardy, R.W. (Eds.), Fish Nutrition, third edition, Academic Press, San Diego, CA, pp. 143 – 179. Zaugg, W.S., McLain, L.R., 1969. Inorganic salt effects on growth, salt water adaptation and gill ATPase of Pacific salmon. In: Halver, J.E., Neuhaus, O.W. (Eds.), Fish in Research. Academic Press, New York, NY, pp. 293 – 306. Zaugg, W.S., Roley, D.D., Prentice, E.F., Gores, K.X., Waknitz, F.W., 1983. Increased seawater survival and contribution to the fishery of chinook salmon (Oncorhynchus tshawytscha) by supplemented dietary salt. Aquaculture 32, 183 – 188.
Effects of adding salt to the diet of Asian sea bass Lates calcarifer reared in fresh or salt water recirculating tanks, on growth and brush border enzyme activity Sheenan Harpaza,T, Yaniv Hakima, Tatiyana Slosmana, O. Tufan Eroldogana,b a
Department of Aquaculture, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel b Department of Aquaculture, Faculty of Fisheries, Cukurova University, Bacali-Adana 01330, Turkey Received 15 July 2004; received in revised form 30 September 2004; accepted 1 March 2005
Abstract The Asian sea bass is a carnivorous, euryhaline species originating in seawater. It is currently being reared under intensive conditions in fresh and brackish water recirculating ponds. The effects of feeding sea bass a diet containing different levels of salt (NaCl) were evaluated in closed recirculating tanks in either fresh or salt (20x) water rearing conditions. Under freshwater conditions, the addition of salt to the diet resulted in a significant improvement in the feed conversion ratio (FCR), and had no effect on carcass composition of the fish. Under saltwater (20x) rearing conditions, it had no affect on either growth or FCR. The addition of salt enhanced the activity of the brush border enzymes alkaline phosphatase, lactase, and, to some extent, leucine amino peptidase in fish reared in freshwater, with the most pronounced effect exhibited in the pyloric caeca. The overall activity level of the brush border enzymes of fish reared in saltwater was found to be higher than that of the fish reared in freshwater. Fish reared in saltwater exhibited significant higher enzymatic activity of maltase, sucrase, and g-glutamyl transpeptidase when fed a diet with added salt compared to the control treatment. The results show that the addition of salt to the diet of Asian sea bass reared in freshwater at a level of up to 4% leads to better feed utilization coupled with a reduction in feed cost. D 2005 Elsevier B.V. All rights reserved. Keywords: Lates calcarifer; Asian sea bass; Dietary salt; Brush border enzymes
1. Introduction
T Corresponding author. Tel.: +972 3 9683388; fax: +972 3 9605667. E-mail address: [email protected] (S. Harpaz). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.03.007
The Asian sea bass, also known as Barramundi, is a carnivorous, euryhaline species originating in seawater, but which can be reared in freshwater and at high densities. It can be cultured in a variety of production systems including cages, and flow-through
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and closed recirculating production units. Barramundi are currently being explored as potential candidates for such systems, using marine water, brackish water, or freshwater. In freshwater-adapted fish, the passive outward flux of ions such as Na+ and Cl from the fish to the external medium, via the gills, faeces, and renal system, must be overcome by active uptake of ions (e.g., Na+, Cl, K+, and Ca2+) from the water and/or from the diet (Smith et al., 1989; Schmidt-Nielsen, 1997). Therefore, the diet constitutes an important source of salts that can satisfy the osmoregulatory requirements of fish kept in freshwater and thus spare energy used for osmoregulation, leaving more energy available for growth (Zaugg et al., 1983; Gatlin et al., 1992). It has been shown that the salinity of the rearing water has an influence on feed intake in rainbow trout. In salinities higher than freshwater (up to 28x), feed intake increased but the growth rate decreased, negatively affecting feed conversion ratio (MacLeod, 1977, 1978; Jurss et al., 1985). Yet, in euryhaline species, salinity affects growth in various ways and maximal growth is not always obtained under isosmotic conditions (Brett, 1979). Many researchers have addressed the issue of using dietary salt to help alleviate the problems associated with transferring salmonids to seawater or saltwater growing conditions (Shehadeh and Gordon, 1969; Zaugg and McLain, 1969; Potts et al., 1970; Basulto, 1976; Zaugg et al., 1983; Salman and Eddy, 1990; Pelletier and Besner, 1992). The results of these studies show a marked advantage in the use of dietary salt resulting in better survival. Even the transfer of tilapia, a freshwater (non-anadromous) species, to saltwater (15–20x) conditions was easier for fish that were pre-acclimatized by adding salt to their diet (Fontainhas-Fernandes et al., 2001). Zaugg and McLain (1969) reported that adding salt to the diet of fish has several advantages: it increases appetite and also acts as a humectant by reducing water activity. The affect on fish growth is not clear. Some studies have shown a positive effect (Gatlin et al., 1992; Fontainhas-Fernandes et al., 2000; Nandeesha et al., 2000; Eroldogan, 2003); some have not found this treatment advantageous (Shaw et al., 1975; Murray and Andrews, 1979); and others have reported that growth was hampered (Salman and Eddy, 1988). Zaugg and McLain (1969) found that in young coho
salmon, even the addition of only 1.5% salt resulted in a 7% weight gain reduction. Tacon and De Silva (1983) surveyed the mineral composition of various fish feeds available on the market and found that the content of salt in these feeds was on the average 1.5% but could be as high as 6% especially in the feeds used for early stage fingerling salmon. A large percentage of the dietary salt in commercial feeds for carnivorous fish originates from the fish meal component of the diet (Murray and Andrews, 1979). It is therefore important to take this into account when replacing fish meal with various plant-derived meals, which are not as rich in salt. In the Asian sea bass, the effects of different diet components (protein:energy ratio; carbohydrates) on growth were studied by Catacutan and Coloso (1995, 1997); kinematics of eating by Dowling et al. (2000); and effects of starvation and growth compensation by Tian and Qin (2003). The results of these studies show that the Asian sea bass is very similar to other carnivorous fish in its nutritional requirements. Yet, the effects of salt addition to the diet of this fish have not been studied. Since the Asian sea bass originates in seawater, we decided to explore the advantages of adding salt (NaCl) to the fish diet. Better utilization of the food is dependent on both its digestion and absorption. This process is a result of the total activity of digestive enzymes, including the final stages of digestion and absorption which take place in the brush border section of the intestines. The availability of the enzymes and their ability to transfer the digested food through the epithelial luminal membrane of the intestine at that stage are crucial (Klein et al., 1998). The goals of the present study were: to explore the effects of adding different levels of dietary salt to the extruded pelletized diet feed of Asian sea bass; to monitor the fish growth and feed conversion ratio in both freshwater and saltwater; and to study the effects of these treatments on the activity of the brush border digestion enzymes: sucrase, maltase, lactase, g-glutamyl transpeptidase, and leucine-amino peptidase. This was done in three separate sections of the digestive tract, namely, the pyloric caeca, and the upper and lower intestines. Since the final stages of the digestion and absorption are carried out by these enzymes, it is essential to explore the effects of the different treatments on their activity.
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2. Materials and methods
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adding freshwater or saltwater in the manner described above.
2.1. Experimental tanks and rearing conditions 2.2. Fish Six plastic tanks with conical bottoms, each containing 250 l, were connected to a central biofilter and settling unit of 350 l, through which the water was circulated with the aid of a submersible pump, at a rate equivalent to total tank replacement every 1.5 h. The bio-filter contained small plastic cylinders, 1.5 cm long with a diameter of 1 cm, kept inside plastic net bags to facilitate cleaning/rinsing. The experimental set up had two such units, totaling 12 tanks altogether. The tank water was heated using electric heating elements (2 kW A) immersed in each of the settling tanks and connected to a thermostat to ensure a constant temperature of 30 8C F1 8C throughout the experiment. The tanks were set indoors and the light source was natural photoperiod enhanced with florescent light, providing a light intensity of 1200 lx during the day hours. A belt feeder situated above each tank provided a small shaded area. Water quality parameters were monitored three times a week and did not deviate from standard conditions considered favorable for these fish (i.e., an oxygen level of 90–100% saturation, average ammonia (measured as NH4+) level of 0.42 mg/l, and an average nitrite level of 1 mg/l). In the saltwater phase, the values were higher and reached an average of 0.84 and 7.75 mg/l, respectively. Ammonia and nitrite levels were determined using Aquamerck kits for fresh and saltwater. Each day, the temperature was registered using a submersed maximum/minimum thermometer; dead fish (if occurred) were removed and the settling tank was cleaned by draining its stand pipe, thus allowing new water to compensate for the water loss during this brief flushing (amounting to approximately 2% of the water volume). Every 3–4 days, a more thorough cleaning of the settling tank was carried out as well as flushing of the organic particles from the biofilter. The amount of water replaced during the course of this procedure was roughly 5%. The water replaced during the saltwater phase of the experiment was a mixture of marine salt and freshwater. The salinity level was monitored every 2–3 days using a refractometer and adjusted by
Postlarvae Asian sea bass (Lates calcarifer) were purchased from Thailand, acclimatized to freshwater at an early stage (postlarvae of less than 0.3 g), and reared under indoor conditions at our laboratory. Fish were selected when they reached an average weight of around 110 g. Prior to the beginning of the experiment, the fish were anesthetized by adding clove oil mixed with ethanol to the tank water. Individual numbered tags were inserted into their caudal muscle and they were weighed using an electronic balance. The experimental tanks were each stocked with a total of 10 fish. The average initial weight of the fish participating in the experiment was 107.22F8.44 g and there were no significant differences among the experimental tanks at the onset of the experiment ( P N 0.05). During the freshwater phase, all the fish in each tank were individually weighed and counted once every fortnight. This was done in order to monitor survival and to update the feeding ration, according to the growth rate in each individual tank. In addition, the food ration was adjusted every other week according to predicted growth. At the end of the freshwater phase, the fish were acclimatized to saltwater by increasing the salinity of the rearing water by 2x per day over a period of 10 days. At the beginning of the saltwater (20x) phase, each tank had seven fish with an initial average weight of 182.11F22.70 g. The fish were individually weighed at the beginning and end of this phase and feeding was adjusted every week according to the predicted growth. The fish were weighed using a digital balance (Swiss quality, Precisa 1000-3000 D) after drying them briefly on tissue paper to absorb excess water. 2.3. Treatments tested 2.3.1. Feed preparation Feed was in the form of sinking extruded pellets— 3 mm in diameter, containing 48% protein, 12% fat, 1.6% fiber, and 1.4% NaCl, and manufactured for carnivorous fish by Raanan Marketing (RMC Ltd., Israel). NaCl-enriched diets were prepared by adding
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the NaCl to the pellets (above the basal level) and spray coating them with a mixture of NaCl and poultry oil to prevent leaching of the salt to the water after immersion. This resulted in a proportional reduction of the ration ingredients replaced by the salt. Preliminary evaluation showed that by using this method, the mineral level in the pellets was not significantly reduced following immersion in water for a period comparable to the time the pellets were consumed by the fish. Proximate analysis of the prepared pellets performed before the beginning of the experiment revealed that the level of ash (corresponding to mineral levels) was proportionally higher in the diets that had salt added to them. 2.3.2. Tested diets A. Feed pellets containing no additional NaCl (control = commercial feed used for predatory fish which has 48% protein, 12% fat, 1.6% fiber, and 1.4% NaCl). B. Feed pellets (as in A) containing 1% additional NaCl. C. Feed pellets (as in A) containing 2% additional NaCl. D. Feed pellets (as in A) containing 4% additional NaCl. Each of the above treatments was tested in three replicates and since two groups of tanks were used, each of the treatments was represented in each group of tanks. According to the feeding charts for Asian sea bass similar in size to the ones used in the present study and kept at a temperature of around 30 8CF1 8C, the feeding rate should be around 2% of the biomass per day. At the onset of the experiment, a ration of 1.5% was used and from 1 week onwards, it was updated to the level of 2% of the biomass per day. Fish were fed 7 days a week with the aid of a belt feeder, which delivered the ration over a period of 6 h during the light phase of the day. The duration of the freshwater testing phase was 50 days, and that of the saltwater (20x) one was 25 days. At the end of the experiment, all the fish in all the tanks were individually weighed in order to compare the effects of the treatments on growth, feed conversion ratio (i.e., total feed given divided by weight gain (FCR)), and survival.
Specific growth rate (SGR) was calculated in the following manner: SGR ¼
ln final weight ln initial weight days of growth
100
2.3.3. Body composition analysis Samples were ground using a meat grinder and thoroughly mixed. Samples of each fish were then taken for analysis as follows: the level of protein was determined according to the Kjeldahl procedure and by the 2000 Digestion System (Tekator, Sweden). Fat was determined using a Soxhelt extractor with 95% ethanol. Ash levels were determined using a BIFATherm C-36 oven (BIFA, Ramat Gan, Israel) set at 600 8C. 2.4. Evaluation of brush border enzymes At the end of each phase of the experiment, three fish from each tank were sacrificed using a scalpel to sever the spine. The fish were taken prior to feeding (to ensure empty digestive tract). The entire digestive tract was removed and adipose tissue carefully cleaned off. When necessary, remaining food residues were gently squeezed out. The digestive system was then divided into the following sections: pyloric ceaca, upper intestines, and lower intestines (each comprising about 50% of the intestine). The dissected sections were stored in marked plastic containers in a 20 8C freezer and evaluated later according to Harpaz and Uni (1999) with slight modifications. Prior to evaluation, stored sections were thawed, transferred to glass tubes, and crushed using a homogenizer. Ten milliliters of distilled water was added to every milliliter of tissue and homogenized using a PRO Scientific Inc. model 200 homogenizer at a speed of 5 V. Between each of the different homogenizing processes, the instrument was thoroughly cleaned with distilled water and wiped with tissue paper. The tissues were kept on ice throughout the whole process. A series of preliminary tests was carried out to determine the appropriate dilutions to be used in the evaluation of the enzymatic activity (Felach, 2002).
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The thawed samples were diluted at a ratio of 1:10 and centrifuged with an Eppendorf 5810R centrifuge for 5 min at a speed of 13,500 g and at 4 8C. 2.4.1. Leucine-amino peptidase (LAP) The activity of LAP (LAP; EC 3.4.11.1) was measured using a kit manufactured by SPINREACT (catalog no. 1001250). One hundred and fifty microliters of reagent were added to 10 Al of the diluted homogenate, placed in an ELISA plate, and, after 5 min of incubation at room temperature, the sample results were read at 1 min intervals at a wave length of 405 nm using a Tecan Sunrise ELISA reader. Units of activity were determined using the following equation: LAPðU=mg PÞ ¼ ðð DA=min TV Þ=ðEF SV ÞÞ ð D=PÞ where: DA/min is total increment per minute; TV (160 Al) is the total reaction volume (homogenate + reagent); EF (1.167) is excitation coefficient of p-nitrophenoxide at 405 nm; SV is homogenate (10 Al); D is dilution (160 Al); and P is protein (mg) in the homogenate. 2.4.2. Alkaline phosphatase (AP) The activity of this enzyme (AP; EC 3.1.3.1) was measured using a kit manufactured by ThermoTrace (catalog no. TR11015). In this reaction, p-nitrophenoxide obtained is proportional to the enzymatic activity and has a distinct yellow color that can be quantified (Walsh, 1979). Two hundred microliters of reagent were added to 5 Al of the diluted homogenate placed in an ELISA plate and read as described for LAP. One unit of enzymatic activity was calculated as the quantity required in order to release 1 Amol of the product ( p-nitrophenoxide) per minute per milligram of protein. Units of activity were determined as described for LAP. Units are the same except for the EF coefficient, which is 18.8, the excitation coefficient of p-nitrophenoxide at 405 nm. 2.4.3. c-Glutamyl transpeptidase (c-GT) The activity of this enzyme (g-GT; EC 2.3.2.2) was measured using a kit manufactured by ThermoTrace
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(catalog no. TR19110). Two hundred microliters of reagent were added to 10 Al of the diluted homogenate placed in an ELISA plate, and read as described for LAP. One unit of enzymatic activity was calculated as the quantity required in order to release 1 Amol of the product (5-amino-2-nitrobenzoate) per minute per milligram of protein (Stump, 1955). Units of activity were determined using the same equation as described for LAP except for the EF coefficient, which is 9.5 for the 5-amino-2-nitrobenzoate produced in this reaction at 405 nm. 2.4.4. Sucrase maltase and lactase determination The activity of the enzymes sucrase (EC 3.2.1.48), maltase (EC 3.2.1.20), and lactase (EC 3.2.1.108) was determined using the following substrates: sucrose produced by Fluka (catalog no. 84099), maltose produced by Sigma (catalog no. M-5885), and lactose produced by Fluka (catalog no. 61340). The activity of these enzymes is as follows: sucrase
sucrose Y glucose þ fructose maltase
maltose Y glucose þ glucose lactase
lactose Y glucose þ galactose The presence of glucose, as a measure of enzymatic activity, was then determined using glucose color reagent no. TR15421 (ThermoTrace) and the reaction was compared to a LT00211.01 standard manufactured by the same company. Forty microliters of homogenate was mixed with 40 Al of substrate at a concentration of 0.056 M. The color reaction was then read at 340 nm using an Ultrospec 2000 Pharmacia Biotech spectrophotometer. Protein was determined using a kit based on the Bradford method and manufactured by Bio-Rad (catalog no. 500-0205). The activity was measured by mixing 20 Al of the homogenate with 1 ml of the reagent, and after 5 min at room temperature, the color intensity was read at 595 nm wave length, while comparing it to a standard (2 mg/ml BSA standard; Bio-Rad color reagent no. 500-0206).
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Statistical analysis was carried out using JMP statistical package. One- and two-way ANOVA followed by a Tukey–Kramer HSD test, set at P b 0.05 were conducted.
Table 2 Body composition of Asian sea bass reared in freshwater and fed diet containing varying levels of added salt Body Control 1% added composition (no added salt) salt Ash Protein Lipid
The growth results for both experiments are presented in Table 1. It can be seen that in the freshwater phase, the addition of salt to the diet resulted in a significantly better feed conversion ratio and a higher (yet not significant) growth rate. In the saltwater phase, the addition of salt to the diet did not result in an improvement of growth or feed conversion ratio. The survival rate in the freshwater phase was very high (97%) and all, except for one tank, had 100% survival. Survival in the saltwater phase was lower (90%) and the variability in growth (Table 1) in this phase was much higher. The body composition of the fish reared in freshwater and fed diets containing different levels of salt was not significantly different among the treatments, although there was a trend showing less fat accumulation in the fish fed diets containing additional salt (Table 2). 3.2. Enzymes activity A clear dose–response relationship between the level of added salt and activity level of alkaline phosphates and lactase was found in the pyloric caeca of fish reared in freshwater (Figs. 1 and 2). The Table 1 Results of growth trials in freshwater and saltwater environments, with Asian sea bass fed diets containing different levels of dietary salt Groups
FW
SW
FCR
SGR
FCR
SGR
Control 1% NaCl 2% NaCl 4% NaCl
1.77 F 0.044b 1.61 F 0.030a 1.60 F 0.023a 1.67 F 0.004ab
0.98 F 0.037 1.08 F 0.039 1.09 F 0.027 1.06 F 0.025
1.37 F 0.167 1.60 F 0.170 1.72 F 0.273 1.37 F 0.183
1.19 F 0.125 1.03 F 0.107 0.99 F 0.157 1.17 F 0.135
Values followed by different superscript letters are significantly different ( P b 0.05).
4% added salt
3.98 F 0.27 3.68 F 0.60 3.59 F 0.54 18.84 F 0.42 19.44 F 0.46 18.72 F 0.91 5.82 F 0.58 6.83 F 1.38 6.57 F 1.00
activity of leucine amino peptidase in the fish reared in freshwater was significantly higher in those fed the diet containing 1% additional salt, while the other levels of salt addition did not result in enhanced activity (Fig. 3). Activity of g-glutamyl transpeptidase was not enhanced by the addition of salt to the diet, but the overall activity in the lower intestine of fish reared in saltwater was significantly higher than that found in this section of the intestine in fish reared in freshwater (Fig. 4). In the fish reared in saltwater, the addition of salt significantly enhanced the activity of maltase, sucrase, and g-glutamyl transpeptidase compared to the con-
a LEVEL OF ACTIVITY (U / mgP)
3.1. Growth and survival
CONTROL 8 7 6 5 4 3 2 1 0
1%
2%
4%
a b b c
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
b LEVEL OF ACTIVITY (U / mgP)
3. Results
4.41 F 0.05 18.91 F 0.22 7.97 F 1.42
2% added salt
CONTROL
1%
2%
4%
9 8 7 6 5 4 3 2 1 0
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
Fig. 1. Activity of alkaline phosphatase (AP) measured in the different sections of the digestive tract of Asian sea bass reared in freshwater (a) and in 20x saltwater (b) and fed diets containing no added salt or 1%, 2%, and 4% added salt.
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CONTROL
500
1%
2%
4%
a
400
a 300
ab
200
b 100 0
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
CONTROL
1%
2%
4%
700 600 500 400 300 200 100 0
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
Fig. 2. Activity of lactase measured in different sections of the digestive tract of Asian sea bass reared in freshwater (a) and in 20x saltwater (b) and fed diets containing no added salt or 1%, 2%, and 4% added salt.
trol. The overall activity of the brush border enzymes was significantly higher in these fish compared with those reared in freshwater in all enzymes tested except for lactase and alkaline phosphatase (Table 3).
4. Discussion Smith et al. (1989) showed that the dietary sodium intake of salmonids kept in freshwater increased by about eightfold from winter to summer (equaling the branchial sodium influx). This corresponds to the increase in feeding and shows that almost all the sodium required can be derived from dietary salt. This can therefore be used as a source of salts for the fish kept in freshwater, providing ions which the fish cannot obtain from the hypotonic environment.
a LEVEL OF ACTIVITY (U / mgP)
LEVEL OF ACTIVITY (µ µmol / mg P / Hour)
b
The addition of salt to the diet of freshwater carp at a level of 1.5% resulted in significantly better growth and is in widespread use in India (Nandeesha et al., 2000). In an experiment conducted with juvenile red drum, it was clearly shown that the addition of 2% NaCl to the diet resulted, as was the case in our study, in greater feed efficiency and greater weight gain (Gatlin et al., 1992). In the same experiment, fish kept in brackish water of 5x and fed a salt-enhanced diet showed an increase in weight gain over the basal diet, but this increase was not significant. Tests conducted on red drum kept in seawater showed no advantage to the addition of NaCl at a level of 2% over the basal diet (Gatlin et al., 1992). The absorption of mono-valent ions increases as a result of the adaptation to the hyperosmotic medium (Shehadeh and Gordon, 1969). It seems that even though fish reared in saltwater were exposed to additional salt from their food, this quantity of salt is negligent compared to the overall amount of salt from the environment that the fish
CONTROL
1%
2%
4%
0.15
a
0.12
b 0.09
b
b
a a ab
0.06
b
0.03 0.00
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
b LEVEL OF ACTIVITY (U / mgP)
LEVEL OF ACTIVITY (µmol / mg P / Hour)
a
321
CONTROL
1%
2%
4%
0.25 0.20 0.15 0.10 0.05 0.00
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
Fig. 3. Activity of leucine amino peptidase (LAP) measured in the different sections of the digestive tract of Asian sea bass reared in freshwater (a) and in 20x saltwater (b) and fed diets containing no added salt or 1%, 2%, and 4% added salt.
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LEVEL OF ACTIVITY (U / mgP)
a
CONTROL
2%
4%
0.11 0.09 0.07 0.04 0.02 0.00
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
b LEVEL OF ACTIVITY (U / mgP)
1%
0.11
0.09
0.07
0.04
0.02
0.00
Pyloric
Upper
Lower
DIGESTIVE TRACT SECTION
Fig. 4. Activity of g-glutamyl transpeptidase (g-GT) measured in the different sections of the digestive tract of Asian sea bass reared in freshwater (a) and in 20x saltwater (b) and fed diets containing no added salt or 1%, 2%, and 4% added salt.
absorb through the intestine and body surface (Potts et al., 1970). The results of our experiment show that fish reared in saltwater and fed feed containing additional salt exhibited a slightly lower (yet non significant) growth rate trend compared with the control group that had no added salt to their feed (Table 1). The enzymatic activity in these fish was different than that observed in freshwater (Figs. 1b, 2b, and 3b). Thus, even if the addition of salt did have a slight impact on growth, this effect is reduced due to better utilization of the food, which is manifested by the increase in the enzymatic activity. It should be mentioned that although the addition of salt did not result in better growth, it did not hamper the growth of the fish reared in saltwater. Therefore, it can be considered as advantageous since the diet containing additional salt in our experiment had a reduction in the level of other ingredients (nutrient dilution) corresponding to the level of added salt.
The overall activity of the brush border enzymes in fish reared in saltwater (20x) was found to be significantly higher than that in fish reared in freshwater. This is probably due to the fact that the Asian sea bass is a catadromous fish, which spends a substantial part of its life in marine water. The better enzymatic activity in the fish fed a diet containing added salt can be explained by the absorption mechanism of the end products—glucose and amino acids. Since the glucose and most of the amino acid absorption is depended on the Na+/K+ ATPase pump (Klein et al., 1998), a higher concentration of Na+ in the lumen might lead to a better absorption of carbohydrates and amino acids. Since the enzyme activity might be inhibited by its end product (carbohydrates or amino acids) (De la Fuente et al., 1997), reduction of the end product concentration can lead to better enzymatic activity in the lumen of fish fed feed enhanced with NaCl. Progressive transfer of rainbow trout to seawater resulted in enhancement of intestinal and branchial Na+/K+ ATPase activity (Fuentes et al., 1997). It is possible that the addition of salt to the feed acted in a similar manner as a bprogressiveQ transfer to salt conditions. Fish feed constitutes one of the most expensive components in the rearing of carnivorous fish, and the extremely high protein levels required for these fish (Wilson, 2002) are also the major source of nitrogenous products harmful to fish in closed recirculating systems. Therefore, the fact that the addition of salt to the diet of the fish reared in freshwater resulted in a better FCR is of great importance. The marked increase in the activity of the brush border enzymes, observed in the freshwater phase, especially in the pyloric caeca (Figs. 1 and 2)
Table 3 Overall activity of different brush border enzymes in Asian sea bass reared in freshwater or saltwater (20 x) medium Enzyme evaluated
Freshwater medium Saltwater medium
Maltase 2184.59 F 73.88 Sucrase 377.48 F 11.02 Lactase 225.96 F 23.07 Leucine amino peptidase 0.074 F 0.0027 g-GT 0.031 F 0.0013 Alkaline phosphatase 2.38 F 0.101 T Values are significantly different ( P b 0.05).
2590.67 F 77.70T 452.21 F16.70T 266.45 F 12.99 0.141 F 0.011T 0.04 F 0.0026T 2.52 F 0.359
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might explain the improved results. The pyloric caeca is emerging as one of the important parts of the digestive tract in carnivorous fish especially as far as the final digestion and absorption of food are concerned (Buddington and Diamond, 1987). Interestingly, in the fish reared in saltwater, the activity of g-glutamyl transpeptidase was much higher in the lower intestine compared with the activity exhibited in the fish held in freshwater and in all other sections of the digestive tract (Fig. 4). The lactase found in the fish intestines most probably originated from bacterial sources. The addition of salt to the diet resulted in an increase in the lactase activity level (Fig. 2), probably due to the altered conditions inside the fish reared in freshwater, which were more favorable for the lactase-supplying bacteria. The conditions in saltwater reared fish were not changed by the additional salt in the diet and therefore the activity of this enzyme in the fish reared in saltwater was not higher (Table 3). The significantly better activity of leucine amino peptidase that was found in the treatment of (1%) salt added to the feed compared with the control and the enhancement of the activity of this protease is of special importance since the Asian sea bass is a carnivorous fish. The presence and high level of activity of carbohydrate-related enzymes in the Asian sea bass have been reported (Sabapathy and Teo, 1993). Maltase is required for the breakdown of maltose produced during the hydrolysis of starch and was found in high levels in the pyloric caeca of Asian sea bass (Sabapathy and Teo, 1993). The intensive rearing of fish in closed recirculating systems is costly and calls for methods that will enhance food utilization while efficiently using the biological filter in the recirculating systems. Utilizing a diet enhanced with up to 4% salt has an advantage, as can be seen from the results obtained in the present study. It can lead to better feed utilization under intensive production conditions and can reduce the cost of feed since we are diluting expensive components with a cheap mineral. This simple method can also be applied by farmers and does not require special means. Total salt content in the diet should also be taken into account when using a plant substitute to replace fish meal, which is one of the major contributors of salt to the diet (Murray and Andrews, 1979).
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Acknowledgement This study was funded by a grant obtained from the European Commission (contract no. ICA3-CT2002-10001).
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